**4. Conclusions**

In the last years, the characterization of trypanosomatid proteomes has become an active area of research. Here, we reported a proteomic analysis of *L. infantum*'s (JPCM5 strain) promastigote stage, and it was the first whole proteomic study in this species after the re-sequencing and de novo assembly of its genome in 2017 by González-De la Fuente et al. [13]. In addition, the search of the MS/MS spectra was performed against any possible ORFs larger than 20 triplets that existed in all-six frames from the *L. infantum* genome sequence. As a result, we identified 2352 proteins (Table S17), most of them corresponding to the predicted sequences in current gene annotations (TriTrypDB.org). Comparisons between the results of this study and previous proteomics data derived from promastigote stages in different *Leishmania* species showed a significant level of similarity regarding the type of detected proteins. Nevertheless, this proteomic study showed experimental evidence on the expression in this parasite stage of 123 proteins that were not detected in previous studies; these proteins are listed in Supplementary File, Table S18. In addition, this study allowed for the identification of several PTMs in proteins, such as phosphorylation, methylation, acetylation, glycosylation, and formylation. Finally, this study also allowed for the identification of eight new protein-coding genes and the extension of the ORFs for 34. In conclusion, whole proteomics and genomic studies are inextricable, the results of the former depend on an accurate genome annotation, and a genome cannot be only annotated in an automatics manner. Thus, the proteomics data obtained in this study have allowed for the correction of annotation mistakes, the discovery of new genes, and experimental evidence of the existence of a large number of proteins that had to date been annotated as hypothetical.

All this new information, at the level of individual genes, is already available at Wikidata.org (searchable by the ID gene) and is going to be incorporated in the TriTrypDB database and the Leish-ESP website (http://leish-esp.cbm.uam.es/).

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4425/11/9/1036/s1, Table S1: Proteins detected in *L. infantum* promastigotes whose orthologues in *L. donovani* were found to be only expressed in the amastigote stage, Table S2: Translational machinery components (ribosomal proteins, translation factors and tRNA synthetases), Table S3: Proteins annotated as RNA binding proteins, Table S4: Identified proteins belonging to the pentose phosphate pathway, Table S5: Proteins of the mithocondrial electron transport chain, Table S6: Identified proteins belonging to the proteasome, Table S7: Identified proteins in the categories of chaperones and heat shock proteins, Table S8: Putative glycosomal proteins, Table S9: Identified proteins with predicted molecular weight higher than 200 kDa, Table S10: Identified proteins in *L. infantum* putatively located in the flagellum and/or involved in parasite motility, Table S11: Enzymes putatively involved on post-translational modifications, Table S12: Methylated proteins and amino acids found to be modified, Table S13: N-terminal acetylated proteins and the modified position, Table S14: Formylated proteins and the modified position, Table S15: Identification of novel ORFs based on peptide MS spectra, Table S16: N-terminal extended proteins based on the identification of peptides mapping upstream of the annotated ORFs, Table S17: Experimentally identified proteins in promastigotes of *L. infantum* JPCM5 strain, Table S18; Proteins identified for the first time in experimental proteomes of *Leishmania* promastigotes.

**Author Contributions:** Conceptualization, Á.S., E.M., A.R., A.M., B.A., and J.M.R.; methodology, Á.S., E.M., A.R., E.C., A.M., and S.G.-d.l.F.; formal analysis, Á.S., E.M., A.R., E.C., and S.G.-d.l.F.; data curation, A.S., E.M., S.G.-d.l.F., and J.M.R.; writing—original draft preparation, Á.S.; writing—review and editing, Á.S., A.M., B.A., and J.M.R.; funding acquisition, A.M., B.A., and J.M.R. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by grants (to B.A. and J.M.R.) from Proyecto del Ministerio de Economía, Industria y Competitividad SAF2017-86965-R, and by the Network of Tropical Diseases Research RICET (RD16/0027/0008); both grants are co-funded with FEDER funds. A.S. was funded by a postdoctoral contract from the "Programa de Empleo Juvenil" of the Community of Madrid, Spain, within the European Youth Employment Initiative (YEI). A.M. was funded by project PRB3-ISCIII (supported by grant PT17/0019) of the PE I+D+i 2013-2016, funded by ISCIII and ERDF. The CBMSO receives institutional grants from the Fundación Ramón Areces and from the Fundación Banco de Santander.

**Acknowledgments:** We thank the Genomics and NGS Core Facility at the Centro de Biología Molecular Severo Ochoa (CBMSO, CSIC-UAM) for helping with the bioinformatics analysis. The CBMSO Proteomics Facility is a member of Proteored.

**Conflicts of Interest:** The authors declare no conflict of interest.
